This application claims the benefit of priority from European Application No. 00400467.7, filed Feb. 21, 2000, the specification of which is incorporated herein in its entirety.
The present invention relates to compounds useful in the inhibition of metalloproteinases and in particular to pharmaceutical compositions comprising these, as well as their use.
The compounds of this invention are inhibitors of one or more metalloproteinase enzymes. Metalloproteinases are a superfamily of proteinases (enzymes) whose numbers in recent years have increased dramatically. Based on structural and functional considerations these enzymes have been classified into families and subfamilies as described in N. M. Hooper (1994) FEBS Letters 354:1-6. Examples of metalloproteinases include the matrix metalloproteinases (MMP) such as the collagenases (MMP1, MMP8, MMP13), the gelatinases (MMP2, MMP9), the stromelysins (MMP3, MMP10, MMP11), matrilysin (MMP7), metalloelastase (MMP12), enamelysin (MMP19), the MT-MMPs (MMP14, MMP15, MMP16, MMP17); the reprolysin or adamalysin or MDC family which includes the secretases and sheddases such as TNF converting enzymes (ADAM10 and TACE); the astacin family which include enzymes such as procollagen processing proteinase (PCP); and other metalloproteinases such as aggrecanase, the endothelin converting enzyme family and the angiotensin converting enzyme family.
Metalloproteinases are believed to be important in a plethora of physiological disease processes that involve tissue remodelling such as embryonic development, bone formation and uterine remodelling during menstruation. This is based on the ability of the metalloproteinases to cleave a broad range of matrix substrates such as collagen, proteoglycan and fibronectin. Metalloproteinases are also believed to be important in the processing, or secretion, of biological important cell mediators, such as tumour necrosis factor (TNF); and the post translational proteolysis processing, or shedding, of biologically important membrane proteins, such as the low affinity IgE receptor CD23 (for a more complete list see N. M. Hooper et al., (1997) Biochem J. 321:265-279).
Metalloproteinases have been associated with many disease conditions. Inhibition of the activity of one or more metalloproteinases may well be of benefit in these disease conditions, for example: various inflammatory and allergic diseases such as, inflammation of the joint (especially rheumatoid arthritis, osteoarthritis and gout), inflammation of the gastro-intestinal tract (especially inflammatory bowel disease, ulcerative colitis and gastritis), inflammation of the skin (especially psoriasis, eczema, dermatitis); in tumour metastasis or invasion; in disease associated with uncontrolled degradation of the extracellular matrix such as osteoarthritis; in bone resorptive disease (such as osteoporosis and Paget""s disease); in diseases associated with aberrant angiogenesis; the enhanced collagen remodelling associated with diabetes, periodontal disease (such as gingivitis), corneal ulceration, ulceration of the skin, post-operative conditions (such as colonic anastomosis) and dermal wound healing; demyelinating diseases of the central and peripheral nervous systems (such as multiple sclerosis); Alzheimer""s disease; extracellular matrix remodelling observed in cardiovascular diseases such as restenosis and atheroscelerosis; and chronic obstructive pulmonary diseases, COPD (for example, the role of MMPs such as MMP12 is discussed in Anderson and Shinagawa, 1999, Current Opinion in Anti-inflammatory and Immunomodulatory Investigational Drugs, 1(1):29-38).
A number of metalloproteinase inhibitors are known; different classes of compounds may have different degrees of potency and selectivity for inhibiting various metalloproteinases. We have discovered a new class of compounds that are inhibitors of metalloproteinases and are of particular interest in inhibiting MMP-13, as well as MMP-9. The compounds of this invention have beneficial potency and/or pharmacokinetic properties.
MMP13, or collagenase 3, was initially cloned from a cDNA library derived from a breast tumour [J. M. P. Freije et al. (1994) Journal of Biological Chemistry 269(24):16766-16773]. PCR-RNA analysis of RNAs from a wide range of tissues indicated that MMP13 expression was limited to breast carcinomas as it was not found in breast fibroadenomas, normal or resting mammary gland, placenta, liver, ovary, uterus, prostate or parotid gland or in breast cancer cell lines (T47-D, MCF-7 and ZR75-1). Subsequent to this observation MMP13 has been detected in transformed epidermal keratinocytes [N. Johansson et al., (1997) Cell Growth Differ. 8(2):243-250], squamous cell carcinomas [N. Johansson et al., (1997) Am. J. Pathol. 151(2):499-508] and epidermal tumours [K. Airola et al., (1997) J. Invest. Dermatol. 109(2):225-231]. These results are suggestive that MMP13 is secreted by transformed epithelial cells and may be involved in the extracellular matrix degradation and cell-matrix interaction associated with metastasis especially as observed in invasive breast cancer lesions and in malignant epithelia growth in skin carcinogenesis.
Recent published data implies that MMP13 plays a role in the turnover of other connective tissues. For instance, consistent with MMP13""s substrate specificity and preference for degrading type II collagen [P. G. Mitchell et al., (1996) J. Clin. Invest. 97(3):761-768; V. Knauper et al., (1996) The Biochemical Journal 271:1544-1550], MMP13 has been hypothesised to serve a role during primary ossification and skeletal remodelling [M. Stahle-Backdahl et al., (1997) Lab. Invest. 76(5):717-728; N. Johansson et al., (1997) Dev. Dyn. 208(3):387-397], in destructive joint diseases such as rheumatoid and osteo-arthritis [D. Wernicke et al., (1996) J. Rheumatol. 23:590-595, P. G. Mitchell et al., (1996) J. Clin. Invest. 97(3):761-768; O. Lindy et al., (1997) Arthritis Rheum 40(8):1391-1399]; and during the aseptic loosening of hip replacements [S. Imai et al., (1998) J. Bone Joint Surg. Br. 80(4):701-710]. MMP13 has also been implicated in chronic adult periodontitis as it has been localised to the epithelium of chronically inflamed mucosa human gingival tissue [V. J. Uitto et al., (1998) Am. J. Pathol 152(6):1489-1499] and in remodelling of the collagenous matrix in chronic wounds [M. Vaalamo et al., (1997) J. Invest. Dermatol. 109(1):96-101].
MMP9 (Gelatinase B; 92 kDa Type IV Collagenase; 92 kDa Gelatinase) is a secreted protein which was first purified, then cloned and sequenced, in 1989 (S. M. Wilhelm et al (1989) J. Biol Chem. 264 (29):17213-17221. Published erratum in J. Biol Chem. (1990) 265 (36):22570.). A recent review of MMP9 provides an excellent source for detailed information and references on this protease: T. H. Vu and Z. Werb (1998) (In: Matrix Metalloproteinases. 1998. Edited by W. C. Parks and R. P. Mecham. pp115-148. Academic Press. ISBN 0-12-545090-7). The following points are drawn from that review by T. H. Vu and Z. Werb (1998).
The expression of MMP9 is restricted normally to a few cell types, including trophoblasts, osteoclasts, neutrophils and macrophages. However, it""s expression can be induced in these same cells and in other cell types by several mediators, including exposure of the cells to growth factors or cytokines. These are the same mediators often implicated in initiating an inflammatory response. As with other secreted MMPs, MMP9 is released as an inactive Pro-enzyme which is subsequently cleaved to form the enzymatically active enzyme. The proteases required for this activation in vivo are not known. The balance of active MMP9 versus inactive enzyme is further regulated in vivo by interaction with TIMP-1 (Tissue Inhibitor of Metalloproteinases-1), a naturally-occurring protein. TIMP-1 binds to the C-terminal region of MMP9, leading to inhibition of the catalytic domain of MMP9. The balance of induced expression of ProMMP9, cleavage of Pro- to active MMP9 and the presence of TIMP-1 combine to determine the amount of catalytically active MMP9 which is present at a local site. Proteolytically active MMP9 attacks substrates which include gelatin, elastin, and native Type IV and Type V collagens; it has no activity against native Type I collagen, proteoglycans or laminins.
There has been a growing body of data implicating roles for MMP9 in various physiological and pathological processes. Physiological roles include the invasion of embryonic trophoblasts through the uterine epithelium in the early stages of embryonic implantation; some role in the growth and development of bones; and migration of inflammatory cells from the vasculature into tissues. Increased MMP9 expression has observed in certain pathological conditions, thereby implicating MMP9 in disease processed such as arthritis, tumour metastasis, Alzheimer""s, Multiple Sclerosis, and plaque rupture in atherosclerosis leading to acute coronary conditions such as Myocardial Infarction.
WO-99/38843 claims compounds of the general formula
Bxe2x80x94Xxe2x80x94(CH2)mxe2x80x94(CR1R2)nxe2x80x94Wxe2x80x94COY 
for use in the manufacture of a medicament for the treatment or prevention of a condition associated with matrix metalloproteinases. Specifically disclosed is the compound N-{1S-[4-(4-Chlorophenyl) piperazine-1-sulfonylmethyl]-2-methylpropyl}-N-hydroxyformamide.
We have now discovered compounds that are potent MMP13 inhibitors and have desirable activity profiles.
In a first aspect of the invention we now provide compounds of the formula I 
wherein B represents a phenyl group monosubstituted at the 3- or 4-position by halogen or trifluoromethyl, or disubstituted at the 3- and 4-positions by halogen (which may be the same or different); or B represents a 2-pyridyl or 2-pyridyloxy group monosubstituted at the 4-, 5- or 6-position by halogen, trifluoromethyl, cyano or C1-4 alkyl; or B represents a 4-pyrimidinyl group optionally substituted at the 6-position by halogen or C1-4 alkyl;
X represents a carbon or nitrogen atom;
R1 represents a trimethyl-1-hydantoin C2-4alkyl or a trimethyl-3-hydantoin C2-4alkyl group; phenyl or C2-4alkylphenyl monosubstituted at the 3- or 4-position by halogen, trifluoromethyl, thio or C1-3alkyl or C1-3 alkoxy; phenyl-SO2NHC2-4alkyl; 2-pyridyl or 2-pyridyl C2-4alkyl; 3-pyridyl or 3-pyridyl C2-4alkyl; 2-pyrimidine-SCH2CH2; 2- or 4-pyrimidinyl C2-4alkyl optionally monosubstituted by one of halogen, trifluoromethyl, C1-3 alkyl, C1-3 alkyloxy, 2-pyrazinyl optionally substituted by halogen or 2-pyrazinyl C2-4 alkyl optionally substituted by halogen;
Any alkyl groups outlined above may be straight chain or branched.
Preferred compounds of the invention are those wherein any one or more of the following apply:
B represents 4-chlorophenyl, 4-fluorophenyl, 4-bromophenyl or 4-trifluorophenyl; 2-pyridyl or 2-pyridyloxy monosubstituted at the 4- or 5-position such as 5-chloro-2-pyridyl, 5-bromo-2-pyridyl, 5-fluoro-2-pyridyl, 5-trifluoromethyl-2-pyridyl, 5-cyano-2-pyridyl, 5-methyl-2-pyridyl; especially 4-fluorophenyl, 5-chloro-2-pyridyl or 5-trifluoromethyl-2-pyridyl;
X represents a nitrogen atom;
R1 is 3-chlorophenyl, 4-chlorophenyl, 3-pyridyl, 2-pyridylpropyl, 2- or 4-pyrimidinylethyl (optionally monosubstituted by fluorine), 2- or 4-pyrimidinylpropyl, 2-(2-pyrimidinyl)propyl (optionally monosubstitued by fluorine); especially 2-pyrimidinylpropyl, 2-(2-pyrimidinyl)propyl (optionally monosubstitued by fluorine) or 5-fluoro-2-pyrimidinylethyl.
For compounds of formula I, a particular subgroup is represented by compounds wherein B is a phenyl group monosubstituted at the 3- or 4-position by halogen or trifluoromethyl, or disubstituted at the 3- and 4-positions by halogen (which may be the same or different); or B is a 2-pyridyl or 2-pyridyloxy group monosubstituted at the 5- or 6-position by halogen, trifluoromethyl or cyano; or B is a 4-pyrimidinyl group optionally substituted at the 6-position by halogen or C1-4 alkyl; X is a carbon or nitrogen atom; R1 is a trimethyl-1-hydantoin C2-4alkyl or a trimethyl-3-hydantoin C2-4 alkyl group; or R1 is a phenyl or C2-4 alkylphenyl monosubstituted at the 3- or 4-position by halogen, trifluoromethyl, thio or C1-3 alkyl or C1-3 alkoxy; or R1 is phenyl-SO2NHC2-4 alkyl; or R1 is 2-pyridyl or 2-pyridyl C2-4 alkyl; or R1 is 3-pyridyl or 3-pyridyl C2-4 alkyl; or R1 is 2-pyrimidine-SCH2CH2; or R1 is 2- or 4-pyrimidinyl C2-4 alkyl optionally monosubstituted by one of halogen, trifluoromethyl, C1-3 alkyl, C1-3 alkyloxy, 2-pyrazinyl or 2-pyrazinyl C2-4 alkyl; any alkyl group may be straight chain or branched.
It will be appreciated that the particular substituents and number of substituents on B and/or R1 are selected so as to avoid sterically undesirable combinations.
Each exemplified compound represents a particular and independent aspect of the invention.
Where optically active centres exist in the compounds of formula I, we disclose all individual optically active forms and combinations of these as individual specific embodiments of the invention, as well as their corresponding racemates. Racemates may be separated into individual optically active forms using known procedures (cf. Advanced Organic Chemistry: 3rd Edition: author J March, p 104-107) including for example the formation of diastereomeric derivatives having convenient optically active auxiliary species followed by separation and then cleavage of the auxiliary species.
It will be appreciated that the compounds according to the invention can contain one or more asymmetrically substituted carbon atoms. The presence of one or more of these asymmetric centres (chiral centres) in a compound of formula I can give rise to stereoisomers, and in each case the invention is to be understood to extend to all such stereoisomers, including enantiomers and diastereomers, and mixtures including racemic mixtures thereof.
In the examples we disclose the isolation and characterisation of certain enantiomers. Enantiomers may be prepared by the reaction of racemic material with a chiral auxilliary, separation of the diastereomers formed using chromatography, followed by subsequent cleavage of the chiral auxilliary. The diastereomer eluted second from the column (using conditions herein described) and subsequently cleaved gives the more active enantiomer when tested. In each case we believe the active enantiomer has S stereochemistry but do not wish to be limited by this initial determination. The active enantiomer is characterised by its derivative being eluted second from the separation column. Use of different compounds of formula I, alternative columns and/or different solvents may affect the elution order of the most active enantiomer.
In the examples we disclose the isolation and characterisation of certain diastereomers. Chromatographic separation and subsequent testing revealed that the more active diastereomer is eluted first from the separation column (ie the more active diastereomer is characterised by being eluted first from the separation column). Use of different compounds of formula I, alternative columns and/or different solvents may affect the elution order of the most active diastereomer.
For compounds of formula I with two chiral centres we believe the active enantiomer has S,S stereochemistry but do not wish to be limited by this initial determination.
Where tautomers exist in the compounds of formula I, we disclose all individual tautomeric forms and combinations of these as individual specific embodiments of the invention.
As previously outlined the compounds of the invention are metalloproteinase inhibitors, in particular they are inhibitors of MMP13. Each of the above indications for the compounds of the formula I represents an independent and particular embodiment of the invention. Whilst we do not wish to be bound by theoretical considerations, the compounds of the invention are believed to show selective inhibition for any one of the above indications relative to any MMP1 inhibitory activity, by way of non-limiting example they may show 100-1000 fold selectivity over any MMP1 inhibitory activity.
Certain compounds of the invention are of particular use as aggrecanase inhibitors ie. inhibitors of aggrecan degradation. Certain compounds of the invention are of particular use as inhibitors of MMP9 and/or MMP12.
The compounds of the invention may be provided as pharmaceutically acceptable salts. These include acid addition salts such as hydrochloride, hydrobromide, citrate and maleate salts and salts formed with phosphoric and sulphuric acid. In another aspect suitable salts are base salts such as an alkali metal salt for example sodium or potassium, an alkaline earth metal salt for example calcium or magnesium, or organic amine salt for example triethylamine.
They may also be provided as in vivo hydrolysable esters. These are pharmaceutically acceptable esters that hydrolyse in the human body to produce the parent compound. Such esters can be identified by administering, for example intravenously to a test animal, the compound under test and subsequently examining the test animal""s body fluids. Suitable in vivo hydrolysable esters for carboxy include methoxymethyl and for hydroxy include formyl and acetyl, especially acetyl.
In order to use a compound of the formula I or a pharmaceutically acceptable salt or in vivo hydrolysable ester thereof for the therapeutic treatment (including prophylactic treatment) of mammals including humans, it is normally formulated in accordance with standard pharmaceutical practice as a pharmaceutical composition.
Therefore in another aspect the present invention provides a pharmaceutical composition which comprises a compound of the formula I or a pharmaceutically acceptable salt or an in vivo hydrolysable ester and pharmaceutically acceptable carrier.
The pharmaceutical compositions of this invention may be administered in standard manner for the disease condition that it is desired to treat, for example by oral, topical, parenteral, buccal, nasal, vaginal or rectal adminstration or by inhalation. For these purposes the compounds of this invention may be formulated by means known in the art into the form of, for example, tablets, capsules, aqueous or oily solutions, suspensions, emulsions, creams, ointments, gels, nasal sprays, suppositories, finely divided powders or aerosols for inhalation, and for parenteral use (including intravenous, intramuscular or infusion) sterile aqueous or oily solutions or suspensions or sterile emulsions.
In addition to the compounds of the present invention the pharmaceutical composition of this invention may also contain, or be co-administered (simultaneously or sequentially) with, one or more pharmacological agents of value in treating one or more disease conditions referred to hereinabove.
The pharmaceutical compositions of this invention will normally be administered to humans so that, for example, a daily dose of 0.5 to 75 mg/kg body weight (and preferably of 0.5 to 30 mg/kg body weight) is received. This daily dose may be given in divided doses as necessary, the precise amount of the compound received and the route of administration depending on the weight, age and sex of the patient being treated and on the particular disease condition being treated according to principles known in the art.
Typically unit dosage forms will contain about 1 mg to 500 mg of a compound of this invention.
Therefore in a further aspect, the present invention provides a compound of the formula I or a pharmaceutically acceptable salt or in vivo hydrolysable ester thereof for use in a method of therapeutic treatment of the human or animal body. In particular we disclose use in the treatment of a disease or condition mediated by MMP13 and/or aggrecanase and/or MMP9 and/or MMP12.
In yet a further aspect the present invention provides a method of treating a metalloproteinase mediated disease condition which comprises administering to a warm-blooded animal a therapeutically effective amount of a compound of the formula I or a pharmaceutically acceptable salt or in vivo hydrolysable ester thereof. Metalloproteinase mediated disease conditions include arthritis (such as osteoarthritis), atherosclerosis, chronic obstructive pulmonary diseases (COPD).
In another aspect the present invention provides a process for preparing a compound of the formula I or a pharmaceutically acceptable salt or in vivo hydrolysable ester thereof which process comprises reacting a compound of the formula II with an appropriate compound of the formula R1CHO to yield an alkene of formula III, which is then converted to a compound of formula IV, which is a precursor to compound I, and optionally thereafter forming a pharmaceutically acceptable salt or in vivo hydrolysable ester of the compound of formula I, as set out below: 
A compound of formula II is conveniently prepared by reacting a compound of formula V with a compound of formula VI, wherein Bxe2x80x2 is a precursor of B and Xxe2x80x2 represents X or a precursor of X or an activated form of X suitable for reaction with Bxe2x80x2. II may also be prepared from compound VII as shown below: 
It will be appreciated that many of the relevant starting materials are commercially available. In addition the following table shows details of aldehyde intermediates and their corresponding registry numbers in Chemical Abstracts.
Aldehydes without Chemical Abstracts Registry Numbers
3-(2-pyrimidyl) propionaldehyde. To a solution of 2-Bromopyrimidine (7.95 g, 0.05 M) in acetonitrile (150 mL) was added propargylalcohol (4.2 g, 0.075 M), bis-(triphenylphosphine)-palladium(II)chloride (750 mg, 1 mM), copper iodide (100 mg, 0.5 mM) and triethylamine (25 mL, 0.25 M) and the mixture was stirred and heated at 70xc2x0 C. for 2 hours. An additional amount of propargyl alcohol (2.1 g, 0.038 M), bis-(triphenylphosphine)-palladium(II)chloride (375 mg, 0.5 mil), and copper iodide (50 mg, 0.25 mil) was then added to the reaction mixture which was stirred and heated at 70xc2x0 C. for an additional 1 hour.
The reaction mixture was evaporated to dryness and the residue which was pre-adsorbed on to silica, chromatographed. Elution with ethyl acetate gave 3-(2-pyrimidyl) prop-2-yn-3-ol as a yellow solid 4.45 g (66%). NMR (CDCl3) 2.9 (1H, t), 4.5 (2H, d), 7.3 (1H, d), 8.8 (2H, t), MS found MH+ 135.
3-(2-pyrimidyl) prop-2-yn-1-ol (4.45 g, 0.033 M) was dissolved in ethyl acetate (140 mL), 10% Pd/C (890 mg) was added and the mixture stirred under an atmosphere of hydrogen for 6 hours. The reaction mixture was filtered through Celite and the filtrate evaporated to give 3-(2-pyrimidyl) propan-1-ol as a yellow oil, 4.15 g (91%). NMR (CDCl3) 2.1 (2H, m), 3.2 (2H, t), 3.8 (2H, t), 7.2 (1H, t), 8.7 (2H, d) MS found MH+139.
3-(2-pyrimidyl) propan-1-ol was oxidized to give 3-(2-pyrimidyl) propionaldehyde using the following Swern conditions. To oxalyl chloride (14.3 ml) dissolved in dichloromethane (700 ml) was added DMSO (21.3 ml), maintaining the temperature below xe2x88x9260xc2x0 C. After 15 minutes the alcohol (20.8 g) dissolved in dichloromethane (20 ml) was slowly added followed 30 minutes later by triethylamine (125 ml). After 15 minutes the reaction mixture was allowed to warm to room temperature when water (100 ml) was added. The solvents were separated and the organic layer was washed with water (3xc3x97150 ml), dried (MgSO4) and evaporated to give an oil which was purified by flash column chromatography eluting with ethyl acetate/methanol (5%) to give the product (8.71 g, 43%) as an oil. NMR CDCl3 3.0 (2H, t), 3.4 (2H, t), 7.1 (1H, t), 8.7 (2H, d), 9.9 (1H, s).
Using the procedure described above the following aldehydes were prepared:
4-(2-pyrimidyl) butyraldehyde by using 3-butyn-1-ol in place of propargylalcohol NMR CDCl3 9.8(1H, s), 8.6 (2H, m), 7.15 (1H, m), 3.0 (2H, m), 2.5 (2H, m), 2.2 (2H, m).
3-(2-pyrazinyl)propionaldehyde by using 2-bromopyrazine in place of 2-bromopyrimidine NMR (d6-DMSO) 9.77 (s, 1H), 8.61 (d, 1H), 8.54 (dd, 1H), 8.46 (d, 1H), 3.10 (t, 2H), 2.92 (t, 2H).
4-(2-pyrazinyl)butyraldehyde by using 2-bromopyrazine in place of 2-bromopyrimidine and 3-butyn-1-ol in place of propargyl alcohol NMR (d6-DMSO) 9.68 (s, 1H), 8.56 (m, 2H), 8.49 (m, 1H), 2.80 (t, 2H), 2.5 (m, 2H), 1.96 (m, 2H).
4-(4-trifluoromethylpyrimidin-2-yl)butanal by using 2-chloro-4-trifluoropyrimidine [CAS registry number 33034-67-2] in place of 2-bromopyrimidine and 3-butyno-1-ol in place of propargyl alcohol 1H NMR (CDCl3): 9.80 (s, 1H), 8.92 (d, 1H, J=5.0 Hz), 7.47 (d, 1H, J=5.0 Hz).3.11 (dd, 2H, J=7.5, 7.5 Hz), 2.60 (dd, 2H, J=6.1, 6.1 Hz), 2.21 (m, 3H).
4-(5-fluoropyrimidin-2-yl)butanal by using 2-chloro-5-fluoro-pyrimidine [CAS registry number 62802-42-0] in place of 2-bromopyrimidine and 3-butyno-1-ol in place of propargyl alcohol 1H NMR (CDCl3): 9.90 (s, 1H), 8.52 (s, 2H, J=5.0 Hz), 7.47, .3.47 (m, 2H), 3.33 (dd, 2H, J=6.8, 6.8 Hz), 3.02 (m, 2H).
4-(4-methoxypyrimidin-2-yl)butanal by using 2-chloro-4-methoxy-pyrimidine [CAS registry number 22536-63-6] in place of 2-bromopyrimidine and 3-butyno-1-ol in place of propargyl alcohol 1H NMR (CDCl3): 9.80 (s, 1H), 8.34 (d, 1H, J=5.0 Hz), 6.55 (d, 1H, J=5.0 Hz), 3.97 (s, 3H), 2.91 (dd, 2H, J=6.8, 6.8 Hz), 2.58 (m, 2H), 2.20 (m, 2H)
4-(5-ethylpyrimidin-2-yl)butanal by using 2-chloro-5-ethyl-pyrimidine [CAS registry number 111196-81-7] in place of 2-bromopyrimidine and 3-butyno-1-ol in place of propargyl alcohol 1H NMR (CDCl3): 9.79 (s, 1H), 8.51 (s, 2H), 2.99 (dd, 2H, J=7.4, 7.4 Hz), 2.54 (m, 4H), 2.17 (p, 1H, J=7.4 Hz), 1.04 (t, 2H, J=7.2 Hz).
5-(2-pyrimidyl)pentanal by using 2-bromopyrimidine and 4-pentyn-1-ol in place of propargul alcohol: NMR (CDCl3) 9.8 (1H, s), 8.65 (2H, m), 7.1 (1H, m), 3.0 (2H, m), 2.5 (2H, m), 1.9 (2H, m), 1.7 (2H, m).
3-(5-bromopyrimidin-2-yl)propanal by using 2-iodo-5-bromopyrimidine in place of 2-bromopyrimidine 1H NMR (CDCl3): 9.90 (s, 1H), 8.70 (s, 2H), 3.30 (dd, 2H), 3.0 (dd, 2H).
4-(4-Pyrimidyl)-butan-1-al. 2,4-Dicloropyrimidine (4.47 g, 0.03 M) was dissolved in triethylamine (250 ml) under argon. (Ph3P)2PdCl2 (420 mg, 0.006 M, CuI (28 mg, 0.00015 M) and 3-butyn-1-ol (2.36 ml, 0.03 M) were added and the mixture was stirred at ambient temperature for 18 hrs. After evaporation to dryness, water (250 ml), was added and extracted with dichloromethane. The combined organic phases were dried and evaporated to dryness. The residual oil was chromatographed, eluting with iso-hexane/ethyl acetate 1:1 to yield 4-(2 chloro-4-pyrimidyl)-3-butyn-1-ol as an oil (3.3 g) NMR (CDCl3) d 8.5, (d 1H); 7.3, (d 1H); 3.9, (t 2H); 2.8, (m 2H); 1.6, (s 1H). Mass Spec found MH+ 183. This material was hydrogenated as described above, but in the presence of 1 equivalent of triethylamine, to give the required saturated alcohol which was oxidised using the previously described Swern oxidation to give the required 4-(4-pyrimidyl)-butan-1-al. NMR CDCl3 d 9.8, (s 1H); 9.1; (s 1H); 8.5, (d 1H); 7.1, (d 1H); 2.8, (t 2H); 2.5, (t 2H); 2.1, (m 2H). Mass spec found MHxe2x88x92 149.
3-(5-Fluoropyrimidin-2-yl)propanal. To a stirred solution of (E)-1-ethoxy-3-(5-fluoropyrimidin-2-yl)prop-2-enyl ethyl ether and (Z)-1-ethoxy-3-(5-fluoropyrimidin-2-yl)prop-2-enyl ethyl ether (9.7 g, 43 mmol) in dry ethanol (100 ml) at room temperature under an atmosphere of argon, was added 10% palladium on activated charcoal (1.0 g). The reaction flask was then evacuated and filled with hydrogen gas. The mixture was then stirred for 18 hours at room temperature. The reaction was then filtered through a pad of celite and evaporated under reduced pressure to give a yellow oil (8.7 g, 89%). To a solution of this oil (15 g, 66 mmol) in THF (200 ml) at room temperature was added an aqueous solution of hydrochloric acid (36 ml of a 2 M solution, 72 mmol) and the reaction was stirred at room temperature for 3 hours. The reaction was then diluted with ethyl acetate (100 ml) and the pH of the mixture brought to pH=9 by the addition of aqueous sodium hydrogen carbonate solution (saturated, 100 ml). The layers were then separated and the aqueous phase extracted with ethyl acetate (3xc3x97100 ml). The combined organic extracts were then dried (Na2SO4), filtered and evaporated under reduced pressure to give 3-(5-fluoropyrimidin-2-yl)propanal (16 g) which was used without further purification. 1H NMR (CDCl3): 9.90 (s, 1H), 8.50 (s, 2H), 3.33 (dd, 2H, J=6.9, 6.9 Hz), 3.00 (dd, 2H, J=6.9, 6.9 Hz).
The starting material was obtained by the following method: To a solution of 2-chloro-5-fluoro-pyrimidine [CAS registry number 62802-42-0] (9.0 g, 68 mmol) and 1-tributylstannyl-3,3-diethoxyprop-1-ene (42.8 g, 102 mmol, 5:1 mixture of E:Z isomers) in dry DMF (140 ml) under an atmosphere of dry argon, was added sequentially solid potassium carbonate (9.4 g, 68 mmol), tetraethylammonium chloride (11.2 g, 68 mmol) and bis(triphenylphosphine)palladium(II) chloride (2.4 g, 3.4 mmol). The resulting mixture was then heated to 120xc2x0 C. for 3 hours. The reaction was then cooled to room temperature and was diluted with water (100 ml) and diethyl ether (150 ml). This mixture was then filtered through a pad of celite. The layers were separated and the aqueous phase extracted with diethyl ether (3xc3x97100 ml). The combined organic extracts were then dried (MgSO4), filtered and evaporated under reduced pressure. Flash chromatography (silica gel, 10% ethyl acetate in hexanes) then gave the product as a pale yellow oil and a 3:1 mixture of E:Z isomers (9.7 g, 63%).
E-isomer: 1H NMR (CDCl3): 8.53 (s, 2H), 6.99 (dd, 1H, J=15.4, 4.1 Hz), 6.86 (d, 1H, J=15.4 Hz), 5.14 (d, 1H, J=4.1 Hz), 3.56 (m, 4H), 1.24 (t, 6H, J=7.1 Hz)
Z-isomer: 1H NMR (CDCl3): 8.57 (s, 2H), 6.65 (d, 1H, J=12.1 Hz), 6.49 (d, 1H, J=7.5 Hz), 6.09 (dd, 1H, J=12.1, 7.5 Hz), 3.70 (m, 4H), 1.21 (t, 6H, J=7.1 Hz)
An analogous method was used to prepare the following aldehydes using the appropriately substituted 2-chloro-pyrimidine:
3-(4-methoxypyrimidin-2-yl)propanal 1H NMR (CDCl3): 9.82 (s, 1H), 8.34 (d, 1H, J=8.4 Hz), 6.55 (d, 1H, J=7,4 Hz), 3.91 (s, 3H), 3.28 (dd, 2H, J=7.4, 7.4 Hz).2.99 (dd, 2H, J=7.4, 7.4 Hz).
3-(4-trifluoromethylpyrimidin-2-yl)propanal 1H NMR (CDCl3): 9.92 (s, 1H), 8.90 (d, 1H, J=5.0 Hz), 7.47 (d, 1H, J=5.0 Hz), 3.43 (dd, 2H, J=6.8, 6.8 Hz).3.07 (dd, 2H, J=6.8, 6.8 Hz).
3-(5-ethylpyrimidin-2-yl)propanal 1H NMR (CDCl3): 9.91 (s, 1H), 8.49 (s, 2H), 3.31 (dd, 2H, J=6.9, 6.9 Hz).2.98 (dd, 2H, J=6.9, 6.9 Hz), 2.61 (q, 2H, J=7.6 Hz), 1.26 (t, 3H, J=7.6 Hz).
3,5,5-Trimethyl-1-propanal Hydantoin 
A solution of 3,5,5-trimethyl hydantoin [CAS (6345-19-3)] (3.5 g, 0.025 mol), 2-(2-bromoethyl)-1,3-dioxolane (4.8 ml, 0.041 mol), K2CO3 (8.5 g, 0.062 mol), benzyltrimethylammonium chloride (2.23 g, 0.012 mol) in MeCN (100 ml) was refluxed together for 24 hrs. Allowed the reaction to cool to RT and filtered, the filtrate was evaporated in vacuo. The residue was taken into DCM then washed with water (xc3x973), before evaporating in vacuo. The residue was azeotroped with toluene (xc3x973) to afford a yellow oil (5.4 g). The oil was then stirred in THF (30 ml) with conc. HCl (4 ml) at RT for 20 hrs. Neutralised with aqueous NaHCO3 and extracted with DCM (xc3x978). The combined organics were dried over Na2SO4 and evaporated in vacuo to afford a yellow oil (4.3 g) 1H NMR (CDCl3): 9.82 (s, 1H), 3.62 (t, 2H), 3.04 (s, 3H), 2.90 (m, 2H), 1.37 (s, 6H).
1,5,5-Trimethyl-3-propanal Hydantoin 
1,5,5-trimethylhydantoin [CAS (6851-81-6)] (5.0 g, 35.0 mol) was added to a mixture of NaOEt (0.02 g, 0.298 mmol, catalytic) and EtOH (8 ml), and stirred under Argon. The mixture was warmed to 30xc2x0 C. before adding acrolein (2.35 ml) slowly, and the reaction exotherms to 45xc2x0 C. The reaction was allowed to cool to RT and stirred for a further 2 hrs. AcOH (o.136 ml, 2.4 mmol) and silica gel (3.5 g) were added to the mixture before evaporating en vacuo. The product on silica was chromatagraphed on a silica column (eluant: 5% acetone/DCM) to afford a clear oil (6.2 g). Further purification of the residue on alumina (eluant: DCM) afforded a clear oil (2.7 g). 1H NMR (CDCl3): 9.78 (s, 1H), 3.88 (t, 2H), 2.86 (s, 3H), 2.82 (m, 2H), 1.37 (s, 6H).
In an analogous manner 1,5,5-trimethyl-3-butanal hydantoin was prepared [M+H 213].
3-(3-Chlorophenyl)butyraldehyde. A mixture of 3-chloroiodobenzene (2.38 g), palladium acetate (20 mg), sodium bicarbonate (1.01 g) and crotyl alcohol (1.28 ml) in N-methylpyrrolidone (4 ml) was stirred and heated at 130xc2x0 C. for 2 hours. The reaction mixture was allowed to cool, water (50 ml) was added and the mixture was extracted with diethyl ether (2xc3x9750 ml). The combined organic extracts were dried and the residue obtained on removal of the solvent was purified by chromatography through silica eluting with a mixture of ethyl acetate and methylene chloride (1:20) to give the title compound as an oil, yield 519 mg, Mxe2x88x92H=181
3-(2-Pyridyl)butyraldehyde. Prepared by Swern oxidation of the corresponding alcohol (CAS 90642-86-7).
3-(5-Fluoropyrimidin-2-yl)butanal 
Concentrated hydrochloric acid (1 m) was added to a stirring solution of 2-[2-(1,3-dioxolan-2-yl)-1-methylethyl]-5-fluoropyrimidine (1.1 g) in tetrahydrofuran (10 ml) at ambient temperature, stirred for 3 hours then added solid sodium hydrogen carbonate to neutral pH. The mixture was poured onto a Chemelute carrtridge and washed with ethyl acetate (3xc3x9720 ml), the combined organics were dried over Na2SO4 and evaporated in vacuo to afford 3-(5-fluoropyrimidin-2-yl)butanal (300 mg, 35%) which was used without further purification.
The starting material was prepared as follows:
2-[2-(1,3-Dioxolan-2-yl)-1-methylethyl]-5-fluoropyrimidine 
To a stirring suspension of activated xe2x80x9cRiekexe2x80x9d zinc in tetrahydrofuran (21 ml, 1.53 M) was added 2-(2-bromopropyl)-1,3-dioxolane (6.6 g) in tetrahydrofuran (50 ml), a rise in temperature from 21xc2x0 C. to 40xc2x0 C. was observed, heated at 40xc2x0 C. for 1 hour then allowed to cool to ambient temperature before adding 2-chloro-5-fluoropyrimidine (3 g) and [1,2-Bis(diphenylphosphino)-propane]dichloronickel(II) chloride (368 mg). The mixture was stirred at ambient temperature for 4 hours then filtered through a pad of celite and the filtrate evaporated under reduced pressure. Flash chromatography (silica gel, haxane-25% ethyl acetate in hexanes) then gave the product as a pale yellow oil (1.1 g); 1H NMR (d6-DMSO): 8.81 (s, 2H), 4.73 (dd, 1H), 3.66-3.87 (m, 4H), 3.21-3.30 (m, 1H), 2.19 (ddd, 1H), 1.83 (ddd, 1H), 1.27 (d, 3H); m/z 213 (M+1).
2-(2-Bromopropyl)-1,3-dioxolane 
Crotonaldehyde (9.18 g, 108 mmol) was added dropwise to a stirring solution of bromotrimethylsilane (24 g, 156 mmol) at 0xc2x0 C., stirred for 1 hour at 0xc2x0 C. then warmed to room temperature and stirred for a further 1 hour. Ethylene glycol (9.5 g, 156 mmol) and p-tolunesulphonic acid (100 mg) was added and the solution was heated to reflux, water was removed by use of Dean and Stark apparatus. On completion the mixture was cooled to room temperature and washed with aqueous sodiumhydrogen carbonate solution (saturated, 2xc3x9750 ml). The residue was purified by vacuum distillation to give 2-(2-bromopropyl)-1,3-dioxolane (18.8 g, 40-42xc2x0 C. @ 1 mm Hg, 89%)
1H NMR (CDCl3): 5.05 (dd, 1H), 4.18-4.33 (m, 1H), 3.84-4.0 (m, 4H), 2.25 (ddd, 1H), 2.03 (ddd, 1H), 1.75 (d, 3H).
An analogous method was used to prepare the following aldehydes using the appropriately substituted 2-chloro-pyrimidine and 1,3-dioxolane:
3-(5-Chloropyrimidin-2-yl)propanal 
1H NMR (CDCl3): 9.90 (s, 1H), 8.60 (s, 2H), 3.32 (dd, 2H), 3.04 (dd, 2H).
3-(5-Chloropyrimidin-2-yl)butanal 
1H NMR (CDCl3): 9.85 (s, 1H), 8.60 (s, 2H), 3.65 (m, 1H), 3.14 (dd, 1H), 2.75 (dd, 1H), 1.39 (d, 3H).
3-[2-(6-Chloropyrazinyl)]propanal 
3-[2-(6-Chloropyrazinyl)]propanal diethyl acetal (200 mg, 0.82 mmol) was treated with 2 N hydrochloric acid (450 xcexcl) in tetrahydrofuran (2.5 ml) at room temperature for 18 h. After adjusting the pH to 8 using saturated aqueous sodium bicarbonate, the reaction was extracted (xc3x973) with ethyl acetate and the organics dried (anyhdrous sodium sulfate), filtered and concentrated in vacuo to give the title compound as a dark brown oil (137 mg, 98%). This material was used without further purification.
1H NMR (CDCl3) xcex4 9.85 (1H, s); 8.4 (2H, 2 x s); 3.5 (2H, t); 3.0 (2H, t).
The starting material was obtained by the following method:
3-[2-(6-Chloropyrazinyl)]propanal Diethyl Acetal 
3-[2-(6-Chloropyrazinyl)]propynal diethyl acetal, (5.5 g, 22.9 mmol) in ethanol (55 ml) was degassed with argon and platinum (IV) oxide (52 mg, 0.23 mmol) added. The reaction vessel was evacuated and an atmosphere of hydrogen was introduced. After 2 days the reaction mixture was concentrated in vacuo and purified by flash chromatography, eluting with a gradient of 0-50% ethyl acetate in iso-hexane, to give 3-[2-(6-Chloropyrazinyl)]propanal diethyl acetal as a pale yellow oil (1.17 g, 21%).
1H NMR (CDCl3) xcex4 8.4 (1H, s); 8.35 (1H, s); 4.5 (1H, t); 3.75-3.55 (2H, m); 3.55-3.4 (2H, m); 2.9 (2H, dd); 2.1 (2H, dd); 1.2 (6H, t).
3-[2-(6-Chloropyrazinyl)]propynal Diethyl Acetal 
To a solution of 2,6-dichloropyrazine (1 g, 6.7 mmol) and propionaldehyde diethyl acetal (1.1 ml, 7.4 mmol) in acetonitrile (10 ml) at room temperature under an atmosphere of argon was added bis(triphenylphosphine)palladium(II) dichloride (94 mg, 0.13 mmol) and copper (I) iodide (51 mg, 0.27 mmol), followed by triethylamine (4.7 ml, 33.6 mmol). The reaction was stirred at room temperature over night. The solvent was removed in vacuo and the residue purified by flash chromatography, eluting with 10-20% ethyl acetate in iso-hexane, to give 3-[2-(6-Chloropyrazinyl)]propynal diethyl acetal as a yellow oil (660 mg, 41%).
1H NMR (CDCl3) xcex4 8.6 (1H, s); 8.55 (1H, s); 5.5 (1H, s); 3.9-3.75 (2H, m); 3.7-3.4 (2H, m); 1.25 (6H, t)
m/s (EI+) 241/243 (MH+).
An alternative process for preparing a compound of the formula I or a pharmaceutically acceptable salt or in vivo hydrolysable ester thereof comprises reacting a compound of the formula II with a compound of the formula R1COOR to yield a compound of the formula VIII, converting this to a compound of the formula IX, converting the compound of formula IX to an alkene of formula III, which is then converted to a compound of formula IV, which is a precursor to compound I, and optionally thereafter forming a pharmaceutically acceptable salt or in vivo hydrolysable ester of the compound of formula I, as set out below: 
Appropriate esters of the formula R1COOR may be commercially or otherwise available or may be produced using, for example, an analogous procedure to that described in Example 10. It will be appreciated that it is possible to use any ester of the formula R1COOR (wherein R1 is as previously defined):xe2x80x94R may be any group including, for example, alkyl, aralkyl, heteroaryl etc.
The compounds of the invention may be evaluated for example in the following assays:
Isolated Enzyme Assays
Matrix Metalloproteinase Family Including For Example MMP13.
Recombinant human proMMP13 may be expressed and purified as described by Knauper et al. [V. Knauper et al., (1996) The Biochemical Journal 271:1544-1550 (1996)]. The purified enzyme can be used to monitor inhibitors of activity as follows: purified proMMP13 is activated using 1 mM amino phenyl mercuric acid (APMA), 20 hours at 21xc2x0 C.; the activated MMP13 (11.25 ng per assay) is incubated for 4-5 hours at 35xc2x0 C. in assay buffer (0.1 M Tris-HCl, pH 7.5 containing 0.1 M NaCl, 20 mM CaCl2, 0.02 mM ZnCl and 0.05% (w/v) Brij 35 using the synthetic substrate 7-methoxycoumarin-4-yl)acetyl.Pro.Leu.Gly.Leu.N-3-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl.Ala.Arg.NH2 in the presence or absence of inhibitors. Activity is determined by measuring the fluorescence at xcexex 328 nm and xcexem 393 nm. Percent inhibition is calculated as follows: % Inhibition is equal to the [Fluorescenceplus inhibitorxe2x88x92Fluorescencebackground] divided by the [Fluorescenceminus inhibitorxe2x88x92Fluorescencebackground].
A similar protocol can be used for other expressed and purified pro MMPs using substrates and buffers conditions optimal for the particular MMP, for instance as described in C. Graham Knight et al., (1992) FEBS Lett. 296(3):263-266.
Adamalysin Family Including for Example TNF Convertase
The ability of the compounds to inhibit proTNFxcex1 convertase enzyme may be assessed using a partially purified, isolated enzyme assay, the enzyme being obtained from the membranes of THP-1 as described by K. M. Mohler et al., (1994) Nature 370:218-220. The purified enzyme activity and inhibition thereof is determined by incubating the partially purified enzyme in the presence or absence of test compounds using the substrate 4xe2x80x2,5xe2x80x2-Dimethoxy-fluoresceinyl Ser.Pro.Leu.Ala.Gln.Ala.Val.Arg.Ser.Ser.Ser.Arg.Cys(4-(3-succinimid-1-yl)-fluorescein)-NH2 in assay buffer (50 mM Tris HCl, pH 7.4 containing 0.1% (w/v) Triton X-100 and 2 mM CaCl2), at 26xc2x0 C. for 18 hours. The amount of inhibition is determined as for MMP13 except xcexex 490 nm and xcexem 530 nm were used. The substrate was synthesised as follows. The peptidic part of the substrate was assembled on Fmoc-NH-Rink-MBHA-polystyrene resin either manually or on an automated peptide synthesiser by standard methods involving the use of Fmoc-amino acids and O-benzotriazol-1-yl-N,N,Nxe2x80x2,Nxe2x80x2-tetramethyluronium hexafluorophosphate (HBTU) as coupling agent with at least a 4- or 5-fold excess of Fmoc-amino acid and HBTU. Ser1 and Pro2 were double-coupled. The following side chain protection strategy was employed; Ser1(But), Gln5(Trityl), Arg8,12(Pmc or Pbf), Ser9,10,11(Trityl), Cys13(Trityl). Following assembly, the N-terminal Fmoc-protecting group was removed by treating the Fmoc-peptidyl-resin with in DMF. The amino-peptidyl-resin so obtained was acylated by treatment for 1.5-2 hr at 70xc2x0 C. with 1.5-2 equivalents of 4xe2x80x2,5xe2x80x2-dimethoxy-fluorescein-4(5)-carboxylic acid [Khanna and Ullman, (1980) Anal Biochem. 108:156-161) which had been preactivated with diisopropylcarbodiimide and 1-hydroxybenzotriazole in DMF]. The dimethoxyfluoresceinyl-peptide was then simultaneously deprotected and cleaved from the resin by treatment with trifluoroacetic acid containing 5% each of water and triethylsilane. The dimethoxyfluoresceinyl-peptide was isolated by evaporation, trituration with diethyl ether and filtration. The isolated peptide was reacted with 4-(N-maleimido)-fluorescein in DMF containing diisopropylethylamine, the product purified by RP-HPLC and finally isolated by freeze-drying from aqueous acetic acid. The product was characterised by MALDI-TOF MS and amino acid analysis.
Natural Substrates
The activity of the compounds of the invention as inhibitors of aggrecan degradation may be assayed using methods for example based on the disclosures of E. C. Arner et al., (1998) Osteoarthritis and Cartilage 6:214-228; (1999) Journal of Biological Chemistry, 274 (10), 6594-6601 and the antibodies described therein. The potency of compounds to act as inhibitors against collagenases can be determined as described by T. Cawston and A. Barrett (1979) Anal. Biochem. 99:340-345.
Inhibition of Metalloproteinase Activity in Cell/Tissue Based Activity Test as an Agent to Inhibit Membrane Sheddases Such as TNF Convertase
The ability of the compounds of this invention to inhibit the cellular processing of TNFxcex1 production may be assessed in THP-1 cells using an ELISA to detect released TNF essentially as described K. M. Mohler et al., (1994) Nature 370:218-220. In a similar fashion the processing or shedding of other membrane molecules such as those described in N. M. Hooper et al., (1997) Biochem. J. 321:265-279 may be tested using appropriate cell lines and with suitable antibodies to detect the shed protein.
Test as an Agent to Inhibit Cell Based Invasion
The ability of the compound of this invention to inhibit the migration of cells in an invasion assay may be determined as described in A. Albini et al., (1987) Cancer Research 47:3239-3245.
Test as an Agent to Inhibit Whole Blood TNF Sheddase Activity
The ability of the compounds of this invention to inhibit TNFxcex1 production is assessed in a human whole blood assay where LPS is used to stimulate the release of TNFxcex1. Heparinized (10 Units/ml) human blood obtained from volunteers is diluted 1:5 with medium (RPMI1640+bicarbonate, penicillin, streptomycin and glutamine) and incubated (160 xcexcl) with 20 xcexcl of test compound (triplicates), in DMSO or appropriate vehicle, for 30 min at 37xc2x0 C. in a humidified (5% CO2/95% air) incubator, prior to addition of 20 xcexcl LPS (E. coli. 0111:B4; final concentration 10 xcexcg/ml). Each assay includes controls of diluted blood incubated with medium alone (6 wells/plate) or a known TNFxcex1 inhibitor as standard. The plates are then incubated for 6 hours at 37xc2x0 C. (humidified incubator), centrifuged (2000 rpm for 10 min; 4xc2x0 C.), plasma harvested (50-100 xcexcl) and stored in 96 well plates at xe2x88x9270xc2x0 C. before subsequent analysis for TNFxcex1 concentration by ELISA.
Test as an Agent to Inhibit in Vitro Cartilage Degradation
The ability of the compounds of this invention to inhibit the degradation of the aggrecan or collagen components of cartilage can be assessed essentially as described by K. M. Bottomley et al., (1997) Biochem J. 323:483-488.
Pharmacodynamic Test
To evaluate the clearance properties and bioavailability of the compounds of this invention an ex vivo pharmacodynamic test is employed which utilises the synthetic substrate assays above or alternatively HPLC or Mass spectrometric analysis. This is a generic test which can be used to estimate the clearance rate of compounds across a range of species. Animals (e,g. rats, marmosets) are dosed iv or po with a soluble formulation of compound (such as 20% w/v DMSO, 60% w/v PEG400) and at subsequent time points (e.g. 5, 15, 30, 60, 120, 240, 480, 720, 1220 mins) the blood samples are taken from an appropriate vessel into 10 U heparin. Plasma fractions are obtained following centrifugation and the plasma proteins precipitated with acetonitrile (80% w/v final concentration). After 30 mins at xe2x88x9220xc2x0 C. the plasma proteins are sedimented by centrifugation and the supernatant fraction is evaporated to dryness using a Savant speed vac. The sediment is reconstituted in assay buffer and subsequently analysed for compound content using the synthetic substrate assay. Briefly, a compound concentration-response curve is constructed for the compound undergoing evaluation. Serial dilutions of the reconstituted plasma extracts are assessed for activity and the amount of compound present in the original plasma sample is calculated using the concentration-response curve taking into account the total plasma dilution factor.
In Vivo Assessment
Test as an Anti-TNF Agent
The ability of the compounds of this invention as ex vivo TNFxcex1 inhibitors is assessed in the rat. Briefly, groups of male Wistar Alderley Park (AP) rats (180-210 g) are dosed with compound (6 rats) or drug vehicle (10 rats) by the appropriate route e.g. peroral (p.o.), intraperitoneal (i.p.), subcutaneous (s.c.). Ninety minutes later rats are sacrificed using a rising concentration of CO2 and bled out via the posterior vena cavae into 5 Units of sodium heparin/ml blood. Blood samples are immediately placed on ice and centrifuged at 2000 rpm for 10 min at 4xc2x0 C. and the harvested plasmas frozen at xe2x88x9220xc2x0 C. for subsequent assay of their effect on TNFxcex1 production by LPS-stimulated human blood. The rat plasma samples are thawed and 175 xcexcl of each sample are added to a set format pattern in a 96 U well plate. Fifty xcexcl of heparinized human blood is then added to each well, mixed and the plate is incubated for 30 min at 37xc2x0 C. (humidified incubator). LPS (25 xcexcl; final concentration 10 xcexcg/ml) is added to the wells and incubation continued for a further 5.5 hours. Control wells are incubated with 25 xcexcl of medium alone. Plates are then centrifuged for 10 min at 2000 rpm and 200 xcexcl of the supernatants are transferred to a 96 well plate and frozen at xe2x88x9220xc2x0 C. for subsequent analysis of TNF concentration by ELISA.
Data analysis by dedicated software calculates for each compound/dose:       Percent    ⁢          xe2x80x83        ⁢    inhibition    ⁢          xe2x80x83        ⁢    of    ⁢          xe2x80x83        ⁢    TNF    ⁢          xe2x80x83        ⁢    α    =                    Mean        ⁢                  xe2x80x83                ⁢        TNF        ⁢                  xe2x80x83                ⁢        α        ⁢                  xe2x80x83                ⁢                  (          Controls          )                    -              Mean        ⁢                  xe2x80x83                ⁢        TNF        ⁢                  xe2x80x83                ⁢        α        ⁢                  xe2x80x83                ⁢                  (          Treated          )                xc3x97        100                    Mean      ⁢              xe2x80x83            ⁢      TNF      ⁢              xe2x80x83            ⁢      α      ⁢              xe2x80x83            ⁢              (        Controls        )            
Test as an Anti-Arthritic Agent
Activity of a compound as an anti-arthritic is tested in the collagen-induced arthritis (CIA) as defined by D. E. Trentham et al., (1977) J. Exp. Med. 146,:857. In this model acid soluble native type II collagen causes polyarthritis in rats when administered in Freunds incomplete adjuvant. Similar conditions can be used to induce arthritis in mice and primates.
Test as an Anti-Cancer Agent
Activity of a compound as an anti-cancer agent may be assessed essentially as described in I. J. Fidler (1978) Methods in Cancer Research 15:399-439, using for example the B16 cell line (described in B. Hibner et al., Abstract 283 p75 10th NCI-EORTC Symposium, Amsterdam Jun. 16-19, 1998).
The invention will now be illustrated but not limited by the following Examples: